By P Koupparis
20 August 2003

Ribonucleic acid (RNA) is a less famous analogue of deoxyribonucleic acid (DNA), although it plays an equally important role in the chemistry of life.

Living organisms express DNA's genetic code as proteins. At any one time, a typical human cell expresses anywhere between a few dozen and several thousand genes. Each gene is transcribed onto a section of single-stranded messenger RNA (mRNA) that carries the genetic code from the cell nucleus to protein factories called ribosomes in the cytoplasm. Each protein molecule is assembled from a string of amino acids. Each amino acid is represented by a 3-base-pair sequence of mRNA called a codon.

The scientific understanding of that mechanism has also provided a way to interfere with the process - with potentially disastrous consequences.

Over the course of billions of years, rogue mRNA molecules have evolved into parasitic, self-replicating objects known as viruses. At the same time, the cell evolved various defence mechanisms to detect and destroy viral RNA.

Viral defence systems lie at the heart of the newly discovered RNAi technology. The "i" in RNAi stands for "interference". Scientists have discovered how to hijack the cell's viral defences to destroy ordinary mRNA and thus silence the expression of the underlying genes.

It is relatively easy to synthesise RNA molecules. When the base sequence of one half of a strand is a reverse complimentary image of the other half, the molecule loops back on itself to create double-stranded RNA (dsRNA).

Pieces of dsRNA longer than about 30-base-pairs induce mammalian cells to shut down gene expression while an enzyme called RNAse L destroys all of the RNA within the cell. Known as the interferon response, it often leads to cell death through a process called apoptosis.

However, dsRNA less than 30-base-pairs in length does not induce the interferon response. Instead, an enzyme called Dicer chops dsRNA into staggered 22-base-pair long fragments. The double-strands are prised apart and one strand, now called a short interfering RNA (siRNA) becomes attached to the target site of an RNA-induced silencing complex (RISC).

The activated RISC then acts like a magnet for any mRNA that either exactly or closely matches 19 base pairs of the target siRNA. Both outcomes halt the translation of mRNA into protein. If the match is close, the RISC sticks to the mRNA. If the match is exact an enzyme called Slicer cleaves the mRNA, the halves of which are then released allowing the RISC to continue destroying matching mRNAs for several days.

Synthetic dsRNA molecules that dice into siRNA fragments matching a section of normal mRNA will silence the expression of a specific gene's protein. In other words, one or more genes can be switched off for several days at a time.

That ability makes RNAi the silver bullet of cellular biochemistry. Such a powerful, gene-specific tool has enormous research and therapeutic potential in medicine. But it also has a negative side, one that scientists appear not to have acknowledged yet, in public, at least.

Several methods for delivering and inserting RNAi fragments into cells are currently under development. RNAi "infection" of cells can be achieved simply by feeding lower animals with bacteria genetically engineered to produce suitably coded RNAi molecules. That technique has not been shown to work in mammals (yet), however, RNAi infection has been achieved with modified adenovirus vectors, high-pressure injection and direct absorption of dsRNA encapsulated within 25-nanometre liposome (i.e. fat) globules.

Liposome encapsulated dsRNA molecules can be inserted into human cells by intravenous injection, inhalation or skin contact via a carrier liniment such as DMSO. As for ingestion, modified liposome nano-particles protected against digestion in the stomach are absorbed through the intestinal tract.

With such a wide range of delivery systems, RNAi technology can be weaponised with ease. Its power can be subverted to serve the needs of the silent assassin, the terrorist or the military.

Long dsRNA kills human cells indiscriminately by inducing apoptosis. Short dsRNA can be designed to induce a wide range of effects from the therapeutic to the deadly.

For example, all of the estimated 100,000 human proteins are manufactured from linear sequences of just twenty amino acids. It is highly likely that there are several 19 base-pair siRNA sequences (equivalent to 6-7 amino acid codons) that are common to many proteins.

A few pieces of dsRNA that dice into such siRNA fragments would disable many essential cell functions at once leading to rapid death or disability.

What makes RNAi a perfect candidate for weaponisation is an earlier, closely related technology called anti-sense RNA. The idea behind that was to synthesise strands of RNA with a complementary (i.e. anti-sense) base-pair sequence to that of target mRNA strands. The anti-sense was supposed to bind to the target mRNA thus halting the expression of a particular gene. In practice, voracious RNAse enzymes devoured most of the anti-sense molecules. If any survived to form long dsRNA, the cell was likely to shut down and commit suicide through apoptosis. Anti-sense RNA's therapeutic promise failed to materialise, although it played an important role in unravelling the mechanics of RNAi.

Short strands of anti-sense RNA can act as an antidote against harmful siRNAs. Slightly mismatched anti-sense RNA fragments will bind to activated RISCs preventing them from silencing targeted mRNAs.

However, an antidote to a particular siRNA can be created only when its code sequence is known. RNAi is a weapon with a built-in antidote protected by a digital key code.

As if the above attributes of RNAi were not enough to raise concerns, it so happens that retroviruses are little more than self-replicating RNA molecules.

Simple organisms have already been modified to produce billions of custom-designed RNAi molecules. That capability can be packaged into the shell of an adenovirus or incorporated into the self-replicating RNA of a retrovirus. Viral vectors can be designed to kill only the individuals infected or to spread like a plague through a target population killing everyone who comes into contact with an infected individual. A similar antidote vector can imbue the aggressor's population with immunity.

The agent can even be programmed to become toxic only in the presence of a particular chemical, such as one found in a specific diet, or in people taking a specific medication, for example.

All the elements for a powerful, versatile bio-weapon are in place. And much of that technology can be exploited in the proverbial well-equipped kitchen.

RNAi technology can kill people in ways that are currently undetectable. In expert hands, it can kill only those who happen to possess one or more genetic variations or mutations. Ethnic groups, tribes, families or individuals can be targeted with ease. A silver bullet par excellence.

RNAi molecules can be designed to interfere with one or more neuro-transmitters rendering the recipient senseless, docile or even insane. Other RNAi sequences can interfere with motor-neurone synapses, mimicking the effects of nerve agents. Blindness, breathing difficulties, pulmonary oedema, haemorrhage, paralysis, heart, liver or kidney failure, fatigue, insatiable thirst or hunger, disorientation or whatever, just program the RNA synthesiser as required. Death by numbers.

Unlike regular poisons and biological toxins, RNAi molecules are virtually undetectable within living cells already teaming with RNA. Lethal RNAi molecules can be ordered through the mail from hundreds of biochemical supply laboratories. Anyone can purchase an RNA synthesiser or simply create short RNA molecules manually with easily purchased organic compounds and reagents.

RNAi can be as toxic as botulinum or as harmless as sherbet. One day, it may even cure cancer - if we survive that long.

© Copyright P Koupparis 2003. All Rights Reserved.

Everything you need to know about RNAi and gene silencing: http://www.ambion.com/techlib/resources/RNAi/

Comprehensive List of RNAi Publications (updated daily): http://www.orbigen.com/RNAi_Orbigen.html